Abstract This proposal focuses on the mechanisms of integrin activation, which is crucial for basic cellular responses essential for normal physiology and pathologies ranging from thrombosis, inflammation, and vascular dysfunction. Integrins play a key role in angiogenesis, or the formation of new blood vessels from existing vasculature. There is a need to understand how angiogenesis is regulated so that innovative approaches for the control of excessive vascular growth in patients with distinct therapeutic needs (i.e. cancer, age-related macular degeneration and retinopathy of diabetes) can be identified. Our mechanistic focus is on Kindlins, a novel family of intracellular mediators which are required for integrin functions on all cells. Although our previous and preliminary studies demonstrated importance of Kindlins in human pathologies, many Kindlins' functions remain to be identified. Thus, the goal of this project is to further understand the molecular mechanisms and structural requirements underlying integrin-dependent and independent functions of kindlins in endothelial and myeloid cells during vascularization processes. The main hypothesis to be tested is that K3 is required for functions of endothelial and myeloid cells in angiogenesis and many, but not all, of these functions are integrin-dependent. The following Aims are proposed: Aim 1. To determine whether K3- dependent regulation of integrins in endothelial and myeloid cells plays a critical role in vascularization in vivo. We will test the hypothesis that K3-integrin interaction is crucial for the regulation of angiogenesis in vivo by using newly generated K3KI knockin mice (expressing a mutant of K3 that is unable to bind to integrin). We will compare the role for K3 on endothelial to that on myeloid cells. Aim 2. To assess the mechanisms responsible for impaired function of endothelial and myeloid cells from K3KI mice, and perform rescue experiments with K3 and K2. To determine the structural requirement for K3-specific functions in EC. Aim 3. To determine whether and how K3 function in hematopoietic cells is regulated during integrin activation with an emphasis on the regulatory role of K3 phosphorylation.